CAJ | 학술논문

目的：探讨成人T淋巴细胞白血病病毒1型（ HTLV-1病毒） Tax蛋白阳性T细胞TaxP中早期生长反应基因-1（EGR-1）表达调控的机制。方法构建不同长度的EGR-1调控序列报告基因，将其转染TaxP细胞，加入NF-κB抑制剂BAY 11-7082或同等体积的二甲基亚砜（DMSO），24 h后收集细胞检测荧光素酶活性；TaxP细胞上清中加入BAY 11-7082或同等体积的DMSO，24 h后免疫印迹检测EGR-1蛋白表达；将Tax及其突变体M22和M47分别转染293 T细胞，24 h后免疫印迹检测EGR-1蛋白表达。结果成功构建了不同长度及突变体的EGR-1调控序列荧光素酶报告基因；荧光素酶报告基因检测显示：相对于E1、E2而言， E3、DelE、MutE的荧光素酶活性明显降低，加入BAY 11-7082后，E1、E2的荧光素酶活性明显降低（P＜0．01），而E3、DelE、MutE无明显变化；同样地，免疫印迹检测显示加入BAY 10-7082后，EGR-1蛋白表达明显降低；相对于野生型和M47，转染M22突变体后，293 T细胞EGR-1蛋白表达明显降低。结论 NF-κB是Tax蛋白阳性T细胞中EGR-1表达调控的关键核因子。
목적：탐토성인T림파세포백혈병병독1형（ HTLV-1병독） Tax단백양성T세포TaxP중조기생장반응기인-1（EGR-1）표체조공적궤제。방법구건불동장도적EGR-1조공서렬보고기인，장기전염TaxP세포，가입NF-κB억제제BAY 11-7082혹동등체적적이갑기아풍（DMSO），24 h후수집세포검측형광소매활성；TaxP세포상청중가입BAY 11-7082혹동등체적적DMSO，24 h후면역인적검측EGR-1단백표체；장Tax급기돌변체M22화M47분별전염293 T세포，24 h후면역인적검측EGR-1단백표체。결과성공구건료불동장도급돌변체적EGR-1조공서렬형광소매보고기인；형광소매보고기인검측현시：상대우E1、E2이언， E3、DelE、MutE적형광소매활성명현강저，가입BAY 11-7082후，E1、E2적형광소매활성명현강저（P＜0．01），이E3、DelE、MutE무명현변화；동양지，면역인적검측현시가입BAY 10-7082후，EGR-1단백표체명현강저；상대우야생형화M47，전염M22돌변체후，293 T세포EGR-1단백표체명현강저。결론 NF-κB시Tax단백양성T세포중EGR-1표체조공적관건핵인자。
Objective To explore the expression of early growth response gene-1 (EGR-1) in T cells that were positive for Tax protein of human T-cell leukemia virus type 1 (HTLV-1) and its possible reg-ulatory mechanism .Methods A series of expression structures carrying the regulatory elements of EGR-1 in different length and luciferase reporter genes were constructed .TaxP cells were transfected with the con-structs containing reporter genes and cultured with 5μmol/L of NF-κB inhibitor BAY 11-7082 or equal vol-ume of DMSO.After cultured for 24 hours the cells were collected to test the luciferase activity .BAY 11-7082 or equal volume of DMSO was added into the supernatant of TaxP cell culture to test the expression of EGR-1 protein by Western blot after 24 hours of culture .Tax and its mutants M22 and M47 were transfected into 293 T cells respectively to test the expression of EGR-1 protein by Western blot after 24 hours of culture . Results The expression structures carrying the regulatory elements of EGR-1 in different length and their mutants followed by luciferase reporter genes were successfully constructed .The luciferase activity in the cells transfected with the constructs containing the elements E 1 and E2 were higher than that transfected with E3, DelE and MutE, but the reporter gene expressions were decreased with the interference of BAY 11-7082 (P<0.01).However, there were no significant changes with the luciferase activity in the cells transfected by elements E3, DelE and MutE.Western blot analysis indicated that the expression of EGR-1 protein was significantly decreased with the interference of BAY 10-7082 .The expression of EGR-1 protein in M22 mu-tants-transfected 293 T cells were decreased significantly in comparison with those by wild type tax-and M47-transfected cells .Conclusion NF-κB was the key nuclear factor in regulating the expression of EGR-1 pro-tein in Tax-positive T cells .